Anti pp2a

Immunoprecipitation experiments were performed using SureBeads™ Protein A/G Magnetic Beads (BioRad SureBeads™ Protein G #161-4023). Briefly, 100 μl SureBeads were magnetized and washed with a solution of PBS/Tween20 0.1% v/v., then incubated for 30 min with 2 μg anti- PP2A (Genetex # GTX106334, Irvine, CA, USA) and Arginase-1 (Genetex # GTX109242, Irvine, CA, USA) at room temperature. After incubation, beads were washed 3 times with PBS/Tween20 0.1% v/v and the beads/antibody complexes were recovered and incubated with 100 μg of protein extracts for 1 h at room temperature. Proteins were separated by SDS-PAGE followed by immunoblotting on a nitrocellulose membrane (Bio-Rad, Hercules CA, USA). Membrane were incubated with the antibodies anti-HNE (1:2000, Novus Biologicals, Abingdon, UK), and then detected by the peroxidase-conjugated secondary antibody (1:20.000, Bio-Rad, Hercules, CA, USA) with Clarity™ Western ECL Blotting Substrates (Bio-Rad, Hercules, CA, US). Membranes were then acquired with ChemiDoc MP image system (Bio-Rad, Hercules, CA, USA) and analyzed using Image Lab software (Bio-Rad, Hercules, CA, USA).